Lipids are potentially useful as carriers for delivery of therapeutic molecules, particularly for delivery of nucleic acids. Lipids form liposomes, which can encapsulate, complex, or entrap nucleic acid molecules and thereby enhance delivery of this class of therapeutic molecules to target cells upon administration, e.g., intravenously to the circulation. Their usefulness in pharmaceutical compositions is limited by available methods to produce lipid-nucleic acid nanoparticles reproducibly. Various methods have been devised to produce such nanoparticles.
Batzri et al., 1973, Biophys Biochem Acta 298:1015-19, and Kremer et al., 1977, Biochemistry 16:3932-35, describe producing lipid vesicles by dissolving lipids in ethanol and injecting the ethanol solution into an aqueous solution in which the lipids spontaneously form liposomes. Hirota et al., 1999, BioTechniques 27:286-89, describe producing lipid vesicles coated with nucleic acid molecules by dissolving cationic lipids in ethanol and injecting the ethanol solution into an aqueous solution containing the nucleic acid molecules. This method fails to produce liposomes that encapsulate nucleic acid.
Maurer et al. U.S. Pat. No. 7,094,423 describe producing liposomes that encapsulate nucleic acids by first preparing preformed single-walled lipid vesicles in an aqueous solution. The preformed lipid vesicles are prepared by dissolving lipids in ethanol, and injecting the lipid mixture into an aqueous buffer. The process of preparing empty preformed vesicles includes sizing by extrusion. Ethanol is added to the empty preformed vesicles after sizing to destabilize them, and nucleic acids in 40% ethanol is added to the destabilized lipid vesicles. After incubation, the mixture is diafiltered to remove ethanol. Variations in the percentage ethanol, temperature, incubation time, lipid composition, drug/lipid ratio, and initial nucleic acid concentration all influence the encapsulation efficiency and yield of this method. For example, Maurer et al. discloses that entrapment increases with increasing oligonucleotide:lipid ratio, reaching a maximum at more than 0.16 mg antisense oligonucleotide per mg lipid, while producing an increase in the number of larger liposomes and in their polydispersity.
Semple et al. U.S. Pat. No. 6,858,225 produce liposomes encapsulating RNA using an ionizable cationic lipid. The lipid is dissolve in ethanol and is combined with nucleic acids in an aqueous buffer at low pH. The ethanol is removed and the pH is brought to neutral pH to form the liposomes. The resulting liposomes are heterogeneous and require homogenization or extrusion to obtain monodisperse lipid vesicles. Consistent with Maurer et al., Semple et al. disclose that entrapment increases with increasing oligonucleotide:lipid ratio, reaching a maximum at more than 0.16 mg antisense oligonucleotide per mg lipid, while producing an increase in the number of larger liposomes and in their polydispersity.
MacLachlan et al. U.S. Pat. No. 7,901,708 describe producing lipid vesicles encapsulating RNA by mixing lipids dissolved in ethanol with RNA in an aqueous solution in a mixing chamber (T-tube) in which the lipids and RNA are diluted stepwise, thereby substantially instantaneously forming vesicles.
Wheeler et al. US 20100041152 describe producing liposomes encapsulating RNA by dissolving cationic lipids in ethanol and mixing with RNA in 65-85% ethanol to produce a soluble, charge-neutralized complex, adding non-cationic lipids to this complex to form a lipid-nucleic acid mixture, and removing ethanol. The liposomes require homogenization or extrusion to obtain monodisperse lipid vesicles.
There remains an unmet need for a manufacturing method to encapsulate nucleic acids without the need for extensive mechanical processing steps to prepare preformed liposomes and without the need for processing step to reduce lipid-nucleic acid particles to a monodisperse population.